We have developed a simple and rapid system for molecularly cloning unitegrated retroviral DNA directly from eukaryotic cells. This "shuttle" vector was constructed by incorporating both plasmid vector DNA sequences from PBR322 and the origin of DNA replication from polyoma virus into the genome of HT-1 MSV. This system has allowed us to molecularly clone infectious, non-permuted, circular forms of unitegrated retroviral DNA. We believe this system will prove useful in elucidating the mechanisms by which retroviruses integrate into the host genome and thereby incorporate cellular proto-oncogenes into viral genomes.